VCIP135 associates with both the N- and C-terminal regions of p97 ATPase.

Two distinct p97ATPase-mediated membrane fusion pathways are required for Golgi and endoplasmic reticulum (ER) biogenesis, namely, the p97/p47 pathway and the p97/p37 pathway. p97 (VCP)/p47 complex-interacting protein p135 (VCIP135) is necessary for both of these pathways. Although VCIP135 is known to form a complex with p97 in the cytosol, the role of this complex in Golgi and ER biogenesis has remained unclear. In this study, we demonstrated that VCIP135 has two distinct p97-binding sites at its N- and C-terminal regions. In particular, the C-terminal binding site includes the SHP motif, which is also found in other p97-binding proteins, such as p47, p37, and Ufd1. We also clarified that VCIP135 binds to both the N- and C-terminal regions of p97; that is, the N- and C-terminal binding sites in VCIP135 interact with the C- and N-terminal regions of p97, respectively. These two interactions within the complex are synchronously controlled by the nucleotide state of p97. We next generated VCIP135 mutants lacking each of the p97-binding sites to investigate their functions in living cells and clarified that VCIP135 is involved in Golgi and ER biogenesis through its two distinct interactions with p97. VCIP135 is hence a unique p97-binding protein that functions by interacting with both the N-and C-terminal regions of p97, which strongly suggests that it plays crucial roles in p97-mediated events.

p97 and p47 function in membrane tethering in cooperation with FTCD during mitotic Golgi reassembly.

p97ATPase-mediated membrane fusion is required for the biogenesis of the Golgi complex. p97 and its cofactor p47 function in soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) priming, but the tethering complex for p97/p47-mediated membrane fusion remains unknown. In this study, we identified formiminotransferase cyclodeaminase (FTCD) as a novel p47-binding protein. FTCD mainly localizes to the Golgi complex and binds to either p47 or p97 via its association with their polyglutamate motifs. FTCD functions in p97/p47-mediated Golgi reassembly at mitosis in vivo and in vitro via its binding to p47 and to p97. We also showed that FTCD, p47, and p97 form a big FTCD-p97/p47-FTCD tethering complex. In vivo tethering assay revealed that FTCD that was designed to localize to mitochondria caused mitochondria aggregation at mitosis by forming a complex with endogenous p97 and p47, which support a role for FTCD in tethering biological membranes in cooperation with the p97/p47 complex. Therefore, FTCD is thought to act as a tethering factor by forming the FTCD-p97/p47-FTCD complex in p97/p47-mediated Golgi membrane fusion.

Mitotic phosphorylation of VCIP135 blocks p97ATPase-mediated Golgi membrane fusion.

In mammals, the Golgi apparatus is disassembled early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 and p37 phosphorylation on Serine-56 and Threonine-59 result in mitotic inhibition of the p97/p47 and the p97/p37 pathways, respectively [11,14]. In this study, we show another mechanism of mitotic inhibition of p97-mediated Golgi membrane fusion. We clarified that VCIP135, an essential factor in both p97 membrane fusion pathways, is phosphorylated on Threonine-760 and Serine-767 by Cdc2 at mitosis and that this phosphorylated VCIP135 does not bind to p97. An in vitro Golgi reassembly assay revealed that VCIP135(T760E, S767E), which mimics mitotic phosphorylation, caused no cisternal regrowth. Our results indicate that the phosphorylation of VCIP135 on Threonine-760 and Serine-767 inhibits p97-mediated Golgi membrane fusion at mitosis.

VCIP135 deubiquitinase and its binding protein, WAC, in p97ATPase-mediated membrane fusion.

Two distinct p97 membrane fusion pathways are required for Golgi biogenesis: the p97/p47 and p97/p37 pathways. VCIP135 is necessary for both pathways, while its deubiquitinating activity is required only for the p97/p47 pathway. We have now identified a novel VCIP135-binding protein, WAC. WAC localizes to the Golgi as well as the nucleus. In Golgi membranes, WAC is involved in a complex containing VCIP135 and p97. WAC directly binds to VCIP135 and increases its deubiquitinating activity. siRNA experiments revealed that WAC is required for Golgi biogenesis. In an in vitro Golgi reformation assay, WAC was necessary only for p97/p47-mediated Golgi reassembly, but not for p97/p37-mediated reassembly. WAC is hence thought to function in p97/p47-mediated Golgi membrane fusion by activating the deubiquitinating function of VCIP135. We also showed that the two p97 pathways function in ER membrane fusion as well. An in vitro ER reformation assay revealed that both pathways required VCIP135 but not its deubiquitinating activity for their ER membrane fusion. This was consistent with the finding that WAC is unnecessary for p97-mediated ER membrane fusion.

Effects of edaravone on muscle atrophy and locomotor function in patients with ischemic stroke: a randomized controlled pilot study.

BACKGROUND AND OBJECTIVE: Stroke patients with severe leg paralysis are often bedridden in the acute and subacute phase, which increases the risk of disuse muscle atrophy in the chronic phase. The evidence to date indicates that oxidative stress plays an important role in the mechanism of disuse muscle atrophy. Therefore, the aim of this study was to determine if long-term radical scavenger treatment with edaravone following an acute stroke prevents the progression of disuse muscle atrophy and improves leg locomotor function in the chronic phase. METHODS: This randomized controlled pilot study was conducted at 19 acute stroke and rehabilitation centers across Japan. Forty-seven ischemic stroke patients with at least leg motor weakness admitted within 24 hours of onset were randomly assigned to receive continuous intravenous infusions of edaravone 30 mg twice daily for 3 days (short-term group) or 10-14 days (long-term group). The primary endpoints of the study included the degree of leg disuse muscle atrophy, as measured by the percentage change from baseline in femoral muscle circumference 15 cm above the knee, and the improvement in leg locomotor function, as assessed by the maximum walking speed over 10 m, 3 months after the onset of stroke. RESULTS: Three-month follow-up was completed by a total of 41 patients (21 in the short-term group and 20 in the long-term group). On admission, there was no significant difference in the severity of stroke or the grade of leg paresis between the two treatment groups. The grade of disuse muscle atrophy and incidence of gait impairment 3 weeks after stroke onset were also similar between the short- and long-term groups. However, disuse muscle atrophy of the paretic and non-paretic legs was significantly less severe in the long-term versus the short-term treatment group (3.6 ± 5.9% and 1.5 ± 6.0% vs 8.3 ± 5.2% and 5.7 ± 6.4%; p < 0.01 and p < 0.05) 3 months after stroke onset. Additionally, the maximum walking speed over a distance of 10 m was significantly greater in the long-term group (98 ± 67 vs 54 ± 55 cm/sec; p < 0.05). CONCLUSION: Edaravone treatment for up to 14 days suppresses the progression of disuse muscle atrophy and improves leg locomotor function to a greater extent than shorter-term treatment in acute stroke patients. This suggests that the management of stroke may be improved with long-term edaravone therapy by providing myoprotective effects that ameliorate functional outcome in the chronic phase.

Phosphorylation of p37 is important for Golgi disassembly at mitosis.

In mammals, the Golgi apparatus is disassembled at early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 by Cdc2 results in mitotic inhibition of the p97/p47 pathway [11]. In this study, we demonstrate that p37 is phosphorylated on Serine-56 and Threonine-59 by Cdc2 at mitosis, and this phosphorylated p37 does not bind to Golgi membranes. Using an in vitro Golgi reassembly assay, we show that mutated p37(S56D, T59D), which mimics mitotic phosphorylation, does not cause any cisternal regrowth, indicating that p37 phosphorylation inhibits the p97/p37 pathway. Our results demonstrate that p37 phosphorylation on Serine-56 and Threonine-59 is important for Golgi disassembly at mitosis.

Isolation of a point-mutated p47 lacking binding affinity to p97ATPase.

p47, a p97-binding protein, functions in Golgi membrane fusion together with p97 and VCIP135, another p97-binding protein. We have succeeded in creating p47 with a point mutation, F253S, which lacks p97-binding affinity. p47 mapping experiments revealed that p47 had two p97-binding regions and the F253S mutation occurred in the first p97-binding site. p47(F253S) could not form a complex with p97 and did not caused any cisternal regrowth in an in vitro Golgi reassembly assay. In addition, mutation corresponding to the p47 F253S mutation in p37 and ufd1 also abolished their binding ability to p97.

Analysis of nucleotide binding to P97 reveals the properties of a tandem AAA hexameric ATPase.

p97, an essential chaperone in endoplasmic reticulum-associated degradation and organelle biogenesis, contains two AAA domains (D1 and D2) and assembles as a stable hexamer. We present a quantitative analysis of nucleotide binding to both D1 and D2 domains of p97, the first detailed study of nucleotide binding to both AAA domains for this type of AAA+ ATPase. We report that adenosine 5'-O-(thiotriphosphate) (ATPgammaS) binds with similar affinity to D1 and D2, but ADP binds with higher affinity to D1 than D2, offering an explanation for the higher ATPase activity in D2. Stoichiometric measurements suggest that although both ADP and ATPgammaS can saturate all 6 nucleotide binding sites in D1, only 3-4 of the 6 D2 sites can bind ATPgammaS simultaneously. ATPgammaS binding triggers a downstream cooperative conformational change of at least three monomers, which involves conserved arginine fingers and is necessary for ATP hydrolysis.

p37 is a p97 adaptor required for Golgi and ER biogenesis in interphase and at the end of mitosis.

We previously reported that p97/p47-assisted membrane fusion is important for the reassembly of organelles at the end of mitosis, but not for their maintenance during interphase. We have now identified a p97 adaptor protein, p37, which forms a complex with p97 in the cytosol and localizes to the Golgi and ER. siRNA experiments revealed that p37 is required for Golgi and ER biogenesis. Injection of anti-p37 antibodies into cells at different cell cycle stages showed that p37 plays an important role in both Golgi and ER maintenance during interphase as well as in their reassembly at the end of mitosis. In an in vitro Golgi reassembly assay, the p97/p37 complex has membrane fusion activity. In contrast to the p97/p47 pathway, this pathway requires p115-GM130 tethering and SNARE GS15, but not syntaxin5. Interestingly, although VCIP135 is also required, its deubiquitinating activity is unnecessary for p97/p37-mediated activities.

Conformational changes in the AAA ATPase p97-p47 adaptor complex.

The AAA+ATPase p97/VCP, helped by adaptor proteins, exerts its essential role in cellular events such as endoplasmic reticulum-associated protein degradation or the reassembly of Golgi, ER and the nuclear envelope after mitosis. Here, we report the three-dimensional cryo-electron microscopy structures at approximately 20 Angstroms resolution in two nucleotide states of the endogenous hexameric p97 in complex with a recombinant p47 trimer, one of the major p97 adaptor proteins involved in membrane fusion. Depending on the nucleotide state, we observe the p47 trimer to be in two distinct arrangements on top of the p97 hexamer. By combining the EM data with NMR and other biophysical measurements, we propose a model of ATP-dependent p97(N) domain motions that lead to a rearrangement of p47 domains, which could result in the disassembly of target protein complexes.

NSF/SNAPs and p97/p47/VCIP135 are sequentially required for cell cycle-dependent reformation of the ER network.

The endoplasmic reticulum (ER) has a characteristic polygonal structure with hallmark three-way junctions. In a previous paper, we reconstituted the disruption of the pre-existing ER network using mitotic cytosol from HeLa cells in streptolysin O (SLO)-permeabilized CHO-HSP cells (stably expressing GFP-HSP47). In addition, we found that interphase cytosol induced reformation of the disrupted ER network into a continuous network structure. Here, we show that the reformation of the ER network is accomplished through two sequential fusion reactions. The first process is mediated by NSF/alpha and gamma-SNAPs, and involves the generation of typical membranous intermediate structures that connect the disrupted ER tubules. A subsequent fusion is mediated by p97/p47/VCIP135, which has been shown to be required for homotypic fusion events in Golgi cisternae regrowth after mitosis. In addition, we also found that both fusion processes involve the t-SNARE, syntaxin 18.

p97/p47-Mediated biogenesis of Golgi and ER.

In mammalian cells, the Golgi apparatus and endoplasmic reticulum have typical structures during interphase: stacked cisternae located adjacent to the nucleus and a network of interconnected tubules throughout the cytoplasm, respectively. At mitosis their architectures disappear and are reassembled in daughter cells. p97, an AAA-ATPase, mediates membrane fusion and is required for reassembly of these organelles. In the p97-mediated membrane fusion, p47 was identified as an essential cofactor, through which p97 binds to a SNARE, syntaxin5. A second essential cofactor, VCIP135, was identified as a p97/p47/syntaxin5-interacting protein. Several lines of recent evidence suggest that ubiquitination may be implicated in the p97/p47 pathway; p47 binds to monoubiquitinated proteins and VCIP135 shows a deubiquitinating activity in vitro. For the cell-cycle regulation of the p97/p47 pathway, it has been reported that the localization and phosphorylation-dephosphorylation of p47 are crucial. In this review, we describe the components involved in the p97-mediated membrane fusion and discuss the regulation of the fusion pathway.

The maintenance of the endoplasmic reticulum network is regulated by p47, a cofactor of p97, through phosphorylation by cdc2 kinase.

The endoplasmic reticulum (ER) has a characteristic complex polygonal structure with hallmark three-way junctions in many types of cells. To investigate the mechanisms responsible for maintaining the ER network, we established ER disassembly and reassembly assays in semi-intact Chinese hamster ovary (CHO) cells that constitutively expressed heat shock protein-47 fused to the green fluorescent protein (GFP-HSP47) as an ER marker (the cells are referred to as CHO-HSP cells). Using these assays, we found that maintenance of the ER network required cytosol and adenosine triphosphate/guanosine 5'-triphosphate (ATP/GTP) hydrolysis, but not actin filaments or microtubules. We also showed that the ER network was disrupted upon addition of either N-ethylmaleimide-treated cytosol after washing semi-intact cells with high salt solution or mitotic cytosol in nocodazole-treated semi-intact CHO-HSP cells. The disrupted ER network induced by mitotic cytosol was reformed by the addition of interphase cytosol. In addition, we found that p47, a cofactor of p97, was essential for the maintenance of the ER network, and that phosphorylation of p47 by cdc2 kinase resulted in ER network disruption by mitotic cytosol. Taken together, these results imply that the maintenance of the ER network requires a membrane fusion process mediated by p97/p47, and that cell cycle-dependent morphological changes of the ER network are regulated through phosphorylation/dephosphorylation of p47.

Cdc2 kinase-dependent disassembly of endoplasmic reticulum (ER) exit sites inhibits ER-to-Golgi vesicular transport during mitosis.

We observed the disassembly of endoplasmic reticulum (ER) exit sites (ERES) by confocal microscopy during mitosis in Chinese hamster ovary (CHO) cells by using Yip1A fused to green fluorescence protein (GFP) as a transmembrane marker of ERES. Photobleaching experiments revealed that Yip1A-GFP, which was restricted to the ERES during interphase, diffused throughout the ER network during mitosis. Next, we reconstituted mitotic disassembly of Yip1A-GFP-labeled ERES in streptolysin O-permeabilized CHO cells by using mitotic L5178Y cytosol. Using the ERES disassembly assay and the anterograde transport assay of GFP-tagged VSVGts045, we demonstrated that the phosphorylation of p47 by Cdc2 kinase regulates the disassembly of ERES and results in the specific inhibition of ER-to-Golgi transport during mitosis.

Structure, dynamics and interactions of p47, a major adaptor of the AAA ATPase, p97.

p47 is a major adaptor molecule of the cytosolic AAA ATPase p97. The principal role of the p97-p47 complex is in regulation of membrane fusion events. Mono-ubiquitin recognition by p47 has also been shown to be crucial in the p97-p47-mediated Golgi membrane fusion events. Here, we describe the high-resolution solution structures of the N-terminal UBA domain and the central domain (SEP) from p47. The p47 UBA domain has the characteristic three-helix bundle fold and forms a highly stable complex with ubiquitin. We report the interaction surfaces of the two proteins and present a structure for the p47 UBA-ubiquitin complex. The p47 SEP domain adopts a novel fold with a betabetabetaalphaalphabeta secondary structure arrangement, where beta4 pairs in a parallel fashion to beta1. Based on biophysical studies, we demonstrate a clear propensity for the self-association of p47. Furthermore, p97 N binding abolishes p47 self-association, revealing the potential interaction surfaces for recognition of other domains within p97 or the substrate.

Structural basis of the interaction between the AAA ATPase p97/VCP and its adaptor protein p47.

The AAA ATPase p97/VCP is involved in many cellular events including ubiquitin-dependent processes and membrane fusion. In the latter, the p97 adaptor protein p47 is of central importance. In order to provide insight into the molecular basis of p97 adaptor binding, we have determined the crystal structure of p97 ND1 domains complexed with p47 C-terminal domain at 2.9 A resolution. The structure reveals that the p47 ubiquitin regulatory X domain (UBX) domain interacts with the p97 N domain via a loop (S3/S4) that is highly conserved in UBX domains, but is absent in ubiquitin, which inserts into a hydrophobic pocket between the two p97 N subdomains. Deletion of this loop and point mutations in the loop significantly reduce p97 binding. This hydrophobic binding site is distinct from the predicted adaptor-binding site for the p97/VCP homologue N-ethylmaleimide sensitive factor (NSF). Together, our data suggest that UBX domains may act as general p97/VCP/CDC48 binding modules and that adaptor binding for NSF and p97 might involve different binding sites. We also propose a classification for ubiquitin-like domains containing or lacking a longer S3/S4 loop.